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BBA - Bioenergetics (v.1807, #9)

Editorial Board (pp. i).

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: Photoreduction of the bacteriopheophytin electron acceptor by Alexej A. Zabelin; Valentina A. Shkuropatova; Vladimir A. Shuvalov; Anatoly Ya. Shkuropatov (pp. 1013-1021).

Mid-infrared spectral changes associated with the photoreduction of the bacteriopheophytin electron acceptor H_A in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus ( Cfl.) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H_A⁻/H_A FTIR (1800–1200cm−¹) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter ( Rba.) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674cm−¹ in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13¹-keto carbonyl of H_A upon the radical anion H_A⁻ formation, exhibits only a very minor upshift to 1675cm−¹ in Cfl. aurantiacus. In contrast, the absorption band of the 13¹-keto C=O of H_A⁻ is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623cm−¹). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 13¹-keto C=O of H_A with Glu L104 and Gln L143 upon H_A⁻ formation and (iii) a possible influence of the protein dielectric environment on the 13¹-keto C=O stretching frequency of neutral H_A. A conformational heterogeneity of the 13³-ester C=O group of H_A is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.► FTIR spectroscopy studies of the bacteriopheophytin electron acceptor H_A in reaction centers from Cfl. aurantiacus. ► A weak hydrogen bond between the anionic state H_A⁻ and Gln L143 is shown. ► A strengthening of the hydrogen bond in the H_A⁻ state is proposed. ► A possible influence of a dielectric environment on the frequency of the keto carbonyl of neutral H_A is discussed. ► Comparisons are made to purple bacteria and Photosystem II.

Keywords: Abbreviations; ∆Absorbance; light-induced absorbance changes; BChl; bacteriochlorophyll; B; _A; and B; _B; BChls on the A and B-branches, respectively; BPheo; bacteriopheophytin; H; _A; and H; _B; BPheos on the A and B-branches, respectively; Blc; Blastochloris; Cfl.; Chloroflexus; FTIR; Fourier transform infrared; IR; infrared; P; primary electron donor; Pheo; pheophytin; a; Pheo; _D1; redox-active Pheo attached to the D1 protein in PSII; PSII; photosystem II; Q; _A; and Q; _B; primary and secondary quinine acceptors, respectively; RC(s); reaction center(s); Rba.; Rhodobacter; T.; Thermosynechococcus; THF; tetrahydrofuranFTIR spectroscopy; Bacterial reaction center; Hydrogen bonding; Bacteriopheophytin acceptor; Chloroflexus aurantiacus; Rhodobacter sphaeroides

Functional domain size in aggregates of light-harvesting complex II and thylakoid membranes by Petar H. Lambrev; Franz-Josef Schmitt; Sabine Kussin; Max Schoengen; Varkonyi Zsuzsanna Várkonyi; Hans Joachim Eichler; Győző Garab; Gernot Renger (pp. 1022-1031).

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers—phenyl- p-benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ^av were quantified for a range of quencher concentrations. Applying the Stern–Volmer formalism, apparent quenching rate constants k_q were determined from the dependencies on quencher concentration of the ratio τ₀^av/ τ^av, where τ₀^av is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k_q′/ k_q, i.e., the apparent quenching rate constants determined in aggregates (or membranes), k_q′, and in detergent-solubilised LHCII trimers, k_q, respectively. In LHCII macroaggregates, the resulting values for the domain size were 15–30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12–24 LHCII trimers, corresponding to 500–1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment–protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.► The functional domain size of photosynthetic pigment–protein assemblies was measured. ► The domain size in LHCII aggregates comprised 15–30 complexes. ► The domain size in native thylakoid membranes comprised 12–24 complexes. ► The functional domain sizes were much smaller than the physical sizes. ► Light-harvesting efficiency cannot be enhanced by simply enlarging the antenna.

Keywords: Abbreviations; AFM; atomic force microscopy; CD; circular dichroism; DAS; decay-associated spectra; DNB; dinitrobenzene; β-DM; n-; dodecyl-β-maltoside; EET; excitation energy transfer; IRF; instrument response function; LHCII; light-harvesting complex II; PPQ; phenyl-; p; -benzoquinone; PSI; photosystem I; PSII; photosystem II; RC; reaction centreConnectivity; Energy transfer; Light-harvesting complex II; Thylakoid membrane; Time-resolved fluorescence; Fluorescence quenching

Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise by Gert Schansker; Toth Szilvia Z. Tóth; Kovacs László Kovács; Alfred R. Holzwarth; Győző Garab (pp. 1032-1043).

Experiments were carried out to identify a process co-determining with Q_A the fluorescence rise between F₀ and F_M. With 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to −10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of Q_A and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at −60°C where recombination between Q_A⁻ and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the F_M. About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the Q_B-occupancy state and PQ-pool quenching. In both +/−DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when Q_A is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction.► Relaxation of the thermal phase at −60°C is incompatible with a redox reaction. ► Thermal phase=fluorescence yield change driven by light-induced conformational change. ► The conformational change(s) occur(s) both in the presence and absence of DCMU. ► The conformational change(s) relax(es) within 100ms in darkness.

Keywords: Abbreviations; Chl; chlorophyll; DCMU; 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; DF; delayed fluorescence; ETC; electron transport chain; F; ₀; and F; _M; fluorescence intensity measured when all photosystem II reaction centers are open or closed respectively; OJIP-transient; fluorescence induction transient defined by the names of its intermediate steps: O; =; 20; μs, J; =; 3; ms, I; =; 30; ms and P; =; the maximum fluorescence intensity; P680; reaction center pigments of photosystem II; PQ; plastoquinone; PSII and PSI; photosystem II and I, respectively; Q; _A; and Q; _B; primary and secondary quinone electron acceptors of photosystem II respectively; S-states S; _0,; S; ₁; , S; ₂; , S; ₃; and S; ₄; different redox states of the oxygen-evolving complexChl; a; fluorescence; OJIP-transient; Delayed fluorescence; DCMU; Thermal phase; Conformational change

Monomeric RC–LH1 core complexes retard LH2 assembly and intracytoplasmic membrane formation in PufX-minus mutants of Rhodobacter sphaeroides by Peter G. Adams; David J. Mothersole; Irene W. Ng; John D. Olsen; C. Neil Hunter (pp. 1044-1055).

In the model photosynthetic bacterium Rhodobacter sphaeroides domains of light-harvesting 2 (LH2) complexes surround and interconnect dimeric reaction centre–light-harvesting 1–PufX (RC–LH1–PufX) ‘core’ complexes, forming extensive networks for energy transfer and trapping. These complexes are housed in spherical intracytoplasmic membranes (ICMs), which are assembled in a stepwise process where biosynthesis of core complexes tends to dominate the early stages of membrane invagination. The kinetics of LH2 assembly were measured in PufX mutants that assemble monomeric core complexes, as a consequence of either a twelve-residue N-terminal truncation of PufX (PufXΔ12) or the complete removal of PufX (PufX⁻). Lower rates of LH2 assembly and retarded maturation of membrane invagination were observed for the larger and less curved ICM from the PufX⁻ mutant, consistent with the proposition that local membrane curvature, initiated by arrays of bent RC–LH1–PufX dimers, creates a favourable environment for stable assembly of LH2 complexes. Transmission electron microscopy and high-resolution atomic force microscopy were used to examine ICM morphology and membrane protein organisation in these mutants. Some partitioning of core and LH2 complexes was observed in PufX⁻ membranes, resulting in locally ordered clusters of monomeric RC–LH1 complexes. The distribution of core and LH2 complexes in the three types of membrane examined is consistent with previous models of membrane curvature and domain formation (Frese et al., 2008), which demonstrated that a combination of crowding and asymmetries in sizes and shapes of membrane protein complexes drives membrane organisation.Display Omitted► The Rba. sphaeroides PufX⁻ mutant has lowered rates of assembly of the LH2 complex. ► The PufX⁻ LH2⁺ mutant has retarded maturation of intracytoplasmic membranes. ► Larger, less curved membranes are observed in the PufX⁻ LH2⁺ mutant. ► AFM shows locally ordered clusters of monomeric RC-LH1 complexes in PufX⁻ membranes. ► Impaired LH2 assembly is consistent with lack of membrane-bending RC–LH1–PufX dimers.

Keywords: Abbreviations; AFM; atomic force microscopy; BChl(s); bacteriochlorophyll(s); B850; bacteriochlorophyll in light-harvesting 2 complex with maximal absorption at 850; nm; B875; bacteriochlorophyll in light-harvesting 1 complex with maximal absorption at 875; nm; EDTA; ethylenediamine tetraacetic acid; HEPES; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; ICM; intracytoplasmic membrane; LD; linear dichroism; LH1; light-harvesting 1 complex; LH2; light-harvesting 2 complex; NIR; near-infrared; Q-PCR; quantitative real-time polymerase chain reaction; Rba.; Rhodobacter; RC; reaction centre; RC–LH1; reaction centre–light-harvesting 1 complex; Rsp.; Rhodospirillum; Rps.; Rhodopseudomonas; TEM; transmission electron microscopy; UPB; upper pigmented band; WT; wild-type; 2-D; two-dimensional; 3-D; three-dimensional; β-DDM; β-dodecylmaltoglucosideBacterial photosynthesis; Light-harvesting; Membrane protein; Protein assembly; PufX; Atomic force microscopy

Carotenoids are essential for normal levels of dimerisation of the RC–LH1–PufX core complex of Rhodobacter sphaeroides: Characterisation of R-26 as a crtB (phytoene synthase) mutant by Irene W. Ng; Peter G. Adams; David J. Mothersole; Cvetelin Vasilev; Elizabeth C. Martin; Helen P. Lang; Jaimey D. Tucker; C. Neil Hunter (pp. 1056-1063).

Carotenoids play important roles in photosynthesis where they are involved in light-harvesting, in photo-protection and in the assembly and structural stability of light-harvesting and reaction centre complexes. In order to examine the effects of carotenoids on the oligomeric state of the reaction centre–light-harvesting 1 -PufX (RC–LH1–PufX) core complex of Rhodobacter sphaeroides two carotenoid-less mutants, TC70 and R-26, were studied. Detergent fractionation showed that in the absence of carotenoids LH2 complexes do not assemble, as expected, but also that core complexes are predominantly found as monomers, although levels of the PufX polypeptide appeared to be unaffected. Analysis of R-26 membranes by electron microscopy and atomic force microscopy reveals arrays of hexagonally packed monomeric RC–LH1–PufX complexes. Transfer of the crtB gene encoding phytoene synthase to TC70 and R-26 restores the normal synthesis of carotenoids demonstrating that the R-26 mutant of Rba. sphaeroides harbours a mutation in crtB, among its other defects. The transconjugant TC70 and R-26 strains containing crtB had regained their ability to assemble wild-type levels of dimeric RC–LH1–PufX core complexes and normal energy transfer pathways were restored, demonstrating that carotenoids are essential for the normal assembly and function of both the LH2 and RC–LH1–PufX complexes in this bacterial photosystem.► The phytoene synthase gene crtB restores normal carotenoid synthesis to R-26. ► Carotenoid-less RC–LH1–PufX complexes are mostly monomeric. ► AFM shows arrays of hexagonally packed RC–LH1–PufX core monomers in R-26 membranes. ► Transfer of crtB restores RC–LH1–PufX dimer formation and energy transfer pathways.

Keywords: Abbreviations; AFM; atomic force microscopy; BChl(s); bacteriochlorophyll(s); EDTA; ethylenediamine tetraacetic acid; HEPES; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; ICM; intracytoplasmic membrane; LH1; light-harvesting 1 complex; LH2; light-harvesting 2 complex; NIR; near-infrared; Rba.; Rhodobacter; RC; reaction centre; RC–LH1; reaction centre–light-harvesting 1; Rsp.; Rhodospirillum; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis; Tris; Tris(hydroxymethyl)aminomethane; β-DDM; β-dodecylmaltoglucosideBacterial photosynthesis; Light-harvesting; Membrane protein; Photosynthetic membrane; Carotenoid; Atomic force microscopy

Absence of mitochondrial uncoupling protein 3: Effect on thymus and spleen in the fed and fasted mice by Orlagh M. Kelly; Richard K. Porter (pp. 1064-1074).

Mitochondrial uncoupling protein 3 (UCP3) is constitutively expressed in mitochondria from thymus and spleen of mice, and confocal microscopy has been used to visualize UCP3 in situ in mouse thymocytes. UCP3 is present in mitochondria of thymus and spleen up to at least 16weeks after birth, but levels decrease by a half in thymus and a fifth in spleen after three weeks, probably reflecting the suckling to weaning transition. UCP3 protein levels increase ~3-fold in thymus on starvation, but expression levels in spleen were unaffected by starvation. Lack of UCP3 had little effect on thymus mass or thymocyte number. However, lack of UCP3 affected spleen mass and splenocyte number (in the fasted state) and results in reduced CD4+ single positive cell numbers and reduced double negative cells in the thymus, but as a 2-fold increase in the proportion of CD4⁺, CD8⁺ and DP cells in spleen. Starvation attenuates these proportionate differences in the spleen. A lack of UCP3 had no apparent effect on basal oxygen consumption of thymocytes or splenocytes or on oxygen consumption due to mitochondrial proton leak. Splenocytes from UCP3 knock-out mice are also more resistant to apoptosis than those from wild-type mice. Overall we can conclude that UCP3 affects thymocyte and spleen cell profiles in the fed and fasted states.► UCP3 present in thymus and spleen. ► No effect of absence of UCP3 on thymus mass and thymocyte number. ► Absence of UCP3 affected spleen mass and splenocytes number in fasted state. ► Lack of UCP3 increases CD4 and CD8 single positive T-cells in the spleen. ► Lack of UCP3 makes splenocytes more resistant to apoptosis.

Keywords: Abbreviations; BCA; bichinchonic acid; BSA; bovine serum albumin; DP; CD4; ⁺; /CD8; ⁺; double positive; DN; CD4; ⁻; /CD8; ⁻; double negative; DEX; dexamethasone; ECL; enhanced chemiluminescence; EGTA; ethylene glycol-bis(2-aminoethylether)-; N; ,; N; ,; N′; ,; N′; -tetraacetic acid; FBS; fetal bovine serum; FCCP; carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; FITC; fluorescein isothiocyanate; PBS; phosphate buffered saline; SP; single positive; PI; propidium iodide; PDH; pyruvate dehydrogenase; PVDF; polyvinylidene difluoride; R-PE; R-phycoerythrin; RPMI; Roswell Park Memorial Institute; TBS; tris buffered saline; UCP; uncoupling protein; WT; wild-typeMitochondrion; Uncoupling; UCP3; Thymus; Spleen

Inhibition of proton pumping in membrane reconstituted bovine heart cytochrome c oxidase by zinc binding at the inner matrix side by Pietro Luca Martino; Giuseppe Capitanio; Nazzareno Capitanio; Sergio Papa (pp. 1075-1082).

A study is presented on the effect of zinc binding at the matrix side, on the proton pump of purified liposome reconstituted bovine heart cytochrome c oxidase (COV). Internally trapped Zn²⁺ resulted in 50% decoupling of the proton pump at level flow. Analysis of the pH dependence of inhibition by internal Zn²⁺ of proton release in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase indicates that Zn²⁺ suppresses two of the four proton pumping steps in the cycle, those taking place when the 2 OH⁻ produced in the reduction of O₂ at the binuclear center are protonated to 2 H₂O. This decoupling effect could be associated with Zn²⁺ induced conformational alteration of an acid/base cluster linked to heme a₃.► In bovine COX Zn²⁺ binding at the N-side decouples by 50% the proton pump at level flow. ► Zinc binding at the N-side suppresses 2 of the 4 pumping steps of the catalytic cycle. ► Zn²⁺ binding causes depression of redox Bohr protons linked to heme a₃.

Keywords: Abbreviations; COX; purified cytochrome; c; oxidase; COV; cytochrome; c; oxidase reconstituted in phospholipid vesicles; Hepes; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Tris; tris(hydroxymethyl)-aminometane; Mes; 2-morpholinoethanesulfonic acid; EDTA; ethylendiaminetetracetic acid; FIC; potassium ferricyanide; CCCP; carbonyl cyanide 3-chloro-phenylhydrazone; BHM; frozen–thawed beef heart mitochondria; R; fully reduced cytochrome; c; oxidase; O; fully oxidized cytochrome; c; oxidase; MV; mixed valence state; P; _M; peroxy state; F; ferryl stateCytochrome; c; oxidase; Proton pumping; Redox Bohr effect; Allosteric cooperativity; Zinc inhibition

Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects by Ann-Louise Johansson; Suman Chakrabarty; Sioberg Catrine L. Berthold; Hogbom Martin Högbom; Arieh Warshel; Peter Brzezinski (pp. 1083-1094).

Cytochrome c oxidase (Cyt cO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa₃ Cyt cO all protons that are pumped across the membrane, as well as one half of the protons that are used for O₂ reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of Cyt cO at a molecular level are related to the timing of proton transfers from Glu286 to a “pump site” and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type Cyt cO as well as in two structural Cyt cO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O₂ reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy–ferryl,P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl–oxidized,F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler,P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.► Understanding mechanisms of proton transfer in cytochrome c oxidase. ► Electron transfer to the catalytic site is gated by protein structural changes. ► Protein structural changes control proton pumping.

Keywords: Abbreviations; Cyt; c; O; cytochrome; c; oxidase; N; ,; P; -side; negative and positive sides of the membrane; SU; subunit; PLS; proton-loading site; KIE; kinetic isotope effect; PMF; potential of mean force; QCP; quantum classical path; k; _B; Boltzmann constant; h; Planck constant; T; temperature; E; _a; Arrhenius activation energy; M; mass; U; potential used in the classical simulation; F; _qm; transmission factor; λ; the “reorganization energy”; k; _H; ,; k; _D; proton or deuteron-transfer rate constants; Δ; H; ^≠; standard enthalpy of activation; Δ; S; ^≠; standard entropy of activation; Δ; G; ^≠; standard Gibbs energy of activation; Δ; g; ^‡; the quantum mechanical activation barrier; Δ; g; ^≠; activation free energy relative to the minimum of the reactant state; P; number of quasiparticles; Δε; _ba; “energy gap” between the reactant and product states; Z; _q; quantum mechanical partition function; ω; effective vibrational frequency; w; potential of mean force; S; _mm; vibrational overlap integral; R; distance between the proton donor and acceptorRespiration; Electron transfer; Cytochrome; aa; ₃; Membrane protein; Electrostatics; Energy transduction

Uncoupled respiration, ROS production, acute lipotoxicity and oxidative damage in isolated skeletal muscle mitochondria from UCP3-ablated mice by Miranda Nabben; Irina G. Shabalina; Esther Moonen-Kornips; Denis van Beurden; Barbara Cannon; Patrick Schrauwen; Jan Nedergaard; Joris Hoeks (pp. 1095-1105).

The function of uncoupling protein 3 (UCP3) is still not established. Mitochondrial uncoupling, control of ROS production, protection against lipotoxicity and protection against oxidative stress are functions classically discussed. To establish a role for UCP3 in these functions, we have here used UCP3 (−/−) mice, backcrossed for 10 generations on a C57Bl/6 background. In isolated skeletal muscle mitochondria, we examined uncoupled respiration, both unstimulated and in the presence of fatty acids. We did not observe any difference between mitochondria from wildtype and UCP3 (−/−) mice. We measured H₂O₂ production rate and respiration rate under reactive oxygen species-generating conditions (succinate without rotenone) but found no effect of UCP3. We tested two models of acute lipotoxicity—fatty acid-induced oxidative inhibition and fatty acid-induced swelling—but did not observe any protective effect of UCP3. We examined oxidative stress by quantifying 4-hydroxynonenal protein adducts and protein carbonyls in the mitochondria—but did not observe any protective effect of UCP3. We conclude that under the experimental conditions tested here, we find no evidence for the function of UCP3 being basal or induced uncoupling, regulation of ROS production, protection against acute lipotoxicity or protection against oxidative damage.► UCP3 does not in itself innately function as an uncoupling protein ► Superoxide and fatty acids are unable to activate UCP3-mediated mitochondrial uncoupling ► Lack of UCP3 does not lead to oxidative damage

Keywords: Abbreviations; ANT; adenine nucleotide translocase; DNPH; dinitrophenylhydrazine; DTT; dithiothreitol; EDTA; ethylenediamine tetraacetic acid; EGTA; ethyleneglycol tetraacetic acid; FCCP; carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone; GDP; guanosine 5′-diphosphate; HEPES; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; 4-HNE; 4-hydroxynonenal; PMSF; phenyl methyl sulfonyl fluoride; SDS; sodium dodecyl sulfate; PBS; phosphate buffered saline; UCP; uncoupling protein; WT; wildtypeUCP3; Oxidative stress; ROS; Skeletal muscle mitochondria; Oxidative capacity; Fatty acids

OXPHOS susceptibility to oxidative modifications: The role of heart mitochondrial subcellular location by Padrao Ana Isabel Padrão; Rita M.P. Ferreira; Rui Vitorino; Renato M.P. Alves; Maria João Neuparth; José Alberto Duarte; Francisco Amado (pp. 1106-1113).

In cardiac tissue two mitochondria subpopulations, the subsarcolemmal and the intermyofibrillar mitochondria, present different functional emphasis, although limited information exists about the underlying molecular mechanisms. Our study evidenced higher OXPHOS activity of intermyofibrillar compared to subsarcolemmal mitochondria, paralleled by distinct membrane proteins susceptibility to oxidative damage and not to quantitative differences of OXPHOS composition. Indeed, subsarcolemmal subunits of respiratory chain complexes were more prone to carbonylation while intermyofibrillar mitochondria were more susceptible to nitration. Among membrane protein targets to posttranslational modifications, ATP synthase subunits alpha and beta were notoriously more carbonylated in both subpopulations, although more intensely in subsarcolemmal mitochondria. Our data highlight a localization dependence of cardiac mitochondria OXPHOS activity and susceptibility to posttranslational modifications.► IMF presented higher respiratory chain complexes activity than SS mitochondria. ► SS and IMF present distinct OXPHOS susceptibility to oxidation and nitration. ► Increased carbonylation levels are paralleled by decreased OXPHOS activity.

Keywords: Abbreviations; 2D-BN-PAGE; two-dimensional blue native polyacrylamide gel electrophoresis; Anti-DNP; anti-2,4-dinitrophenylhydrazone; Anti-3-NT; anti-3-nitrotyrosine; OXPHOS; oxidative phosphorylation; ETC; electron transport chain; IMF; intermyofibrillar mitochondria; SS; subsarcolemmal mitochondriaIntermyofibrillar mitochondria; Subsarcolemmal mitochondria; 2-D BN-PAGE/MS/MS; 3-Nitrotyrosine; Carbonylation; Respiratory chain complexes

Variation of flux control coefficient of cytochrome c oxidase and of the other respiratory chain complexes at different values of protonmotive force occurs by a threshold mechanism by Giovanni Quarato; Claudia Piccoli; Rosella Scrima; Nazzareno Capitanio (pp. 1114-1124).

The metabolic control analysis was applied to digitonin-permeabilized HepG2 cell line to assess the flux control exerted by cytochrome c oxidase on the mitochondrial respiration. Experimental conditions eliciting different energy/respiratory states in mitochondria were settled. The results obtained show that the mitochondrial electrochemical potential accompanies a depressing effect on the control coefficient exhibited by the cytochrome c oxidase. Both the components of the protonmotive force, i.e. the voltage (ΔΨ_m) and the proton (ΔpH_m) gradient, displayed a similar effect. Quantitative estimation of the ΔΨ_m unveiled that the voltage-dependent effect on the control coefficient of cytochrome c oxidase takes place sharply in a narrow range of membrane potential from 170–180 to 200–210mV consistent with the physiologic transition from state 3 to state 4 of respiration. Extension of the metabolic flux control analysis to the NADH dehydrogenase and bc₁ complexes of the mitochondrial respiratory chain resulted in a similar effect. A mechanistic model is put forward whereby the respiratory chain complexes are proposed to exist in a voltage-mediated threshold-controlled dynamic equilibrium between supercomplexed and isolated states.► MCA of mitochondrial respiration was applied to permeabilized cells. ► The mitochondrial protonmotive force depresses the flux control coefficient of COX. ► A narrow threshold range of the ΔΨmt accounts for its effect on COX. ► The respiratory flux controls of CI and CIII but not of CII respond as CIV to ΔΨmt.

Keywords: Abbreviations; MCA; metabolic control analysis; GRF; glogal respiratory flux; COX-IS; cytochrome c oxidase isolated enzymatic step; C; ^J; v; _,COX; flux control coefficient of COX; C; ^J; v; _,CI; flux control coefficient of complex I; RC; respiratory chain; COX; cytochrome c oxidase; Δμ; _H+; mitochondrial transmembrane electrochemical proton gradient; ΔΨ; _m; mitochondrial transmembrane electrical potential; ΔpH; _m; mitochondrial transmembrane pH gradient; OXPHOS; oxidative phosphorylation; FCCP; carbonylcyanide p-trifluoromethoxyphenylhydrazone; TMRE; tetramethylrhodamine, ethyl ester; UQ; ubiquinone; ROS; reactive oxygen speciesMetabolic control analysis; Oxidative phosphorylation; Mitochondrial membrane potential; Cytochrome c oxidase; Respiratory chain supercomplexes

Mitochondria of highly metastatic breast cancer cell line MDA-MB-231 exhibits increased autophagic properties by Yi-Fang Tu; Benny A. Kaipparettu; Yewei Ma; Lee-Jun C. Wong (pp. 1125-1132).

Autophagy is a cellular housekeeping process that removes damaged or unwanted cellular components and recycles them to build new constituents. It is essential for tumor growth under adverse environment. Mitochondria play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome. To understand the contribution of mitochondria to the regulation of homeostatic autophagy in cancer cells, we used the transmitochondrial cytoplasmic hybrid (cybrid) model. Cybrid system allowed us to compare mitochondria from different cell types including highly metastatic breast cancer cell line MDA-MB-231 (c231), less metastatic breast cancer cell lines: MDA-MB-436 (c436) and MDA-MB-468 (c468), as well as non-cancerous mammary epithelial cell MCF-10A (c10A) in a defined nuclear background. The c231 exhibited lower LC3-II levels but higher ratio of LC3-II/LC3-I than c436, c468 and c10A. In addition, c231 displayed more punctate LC3-positive cells and had lower levels of sequestosome 1 (p62/SQSTM1) than other cybrids. These suggested that mitochondria could contribute to the increased autophagy and autophagic flux in metastatic cancer. This increased autophagy was found to be non-selective autophagy instead of selective mitophagy since LC3 puncta in c231 did not co-localize with mitochondria labeled by Mitotracker red or Tomm 20. The promotion of mitochondrial permeability transition (MPT) in c231 also contributed to increased autophagy. Block of MPT by the inhibition of low-conductance stage of MPT pores resulted in a decrease of LC3 puncta in c231. These results suggested that mitochondria from highly metastatic breast cancer cell line MDA-MB-231 can promote homeostatic autophagy of cancer through opening low-conductance MPT pores.► We used cybrids to study the effect of cancer mitochondria on autophagy. ► Metastatic breast cancer mitochondria showed high LC3-II/LC3-I ratio in cybrids. ► Mitochondria from metastatic breast cancer exhibit increased autophagic flux. ► Metastatic cancer mitochondria promote autophagy by opening low-conductance MPT pores. ► Metastatic breast cancer mitochondria contribute to increased autophagic properties.

Keywords: Keyword; Cancer; Mitochondria; Autophagy; Transmitochondrial cytoplasmic hybrid

The Na⁺-translocating F₁F_O-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus by Noha M. Mesbah; Juergen Wiegel (pp. 1133-1142).

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3–3.9M Na⁺, pH50^°C 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F₁ portion and 3-subunit-F_O portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na⁺ (5mM); hydrolysis activity was inhibited by higher Na⁺ concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH50^°C of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N′-dicylohexylcarbodiimide and exhibited inhibition by both free Mg²⁺ and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na⁺-ion gradient that was sensitive to the Na⁺-ionophore monensin. Analysis of the atp operon showed the presence of the Na⁺-binding motif in the c subunit (Q³³, E⁶⁶, T⁶⁷, T⁶⁸, Y⁷¹), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F₁F_O-ATPase of N. thermophilus is a Na⁺-translocating ATPase used primarily for expelling cytoplasmic Na⁺ that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na⁺-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na⁺ needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.► Characterize F-ATPase of halophilic alkalithermophilic Natranaerobius thermophilus. ► F-type ATPase is Na⁺-coupled, hydrolysis stimulated by Na⁺ ions. ► Maximal ATP hydrolysis activity at high temperature (47°C) and alkaline pH (9.3). ► ATPase functions mainly in hydrolysis direction, very low rates of ATP synthesis. ► ATP hydrolysis activity regulated by untruncated epsilon subunit.

Keywords: Abbreviations; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; DCCD; N,N′; -dicylohexylcarbodiimide; CCCP; carbonyl cyanide; m; -chlorophenylhydrazone; P; _i; inorganic phosphate; OD; optical density; DTT; dithiothreitol; PEG; polyethylene glycol Natranaerobius thermophilus; ATPase; Halophile; Alkalithermophile; Epsilon subunit

The small CAB-like proteins of the cyanobacterium Synechocystis sp. PCC 6803: Their involvement in chlorophyll biogenesis for Photosystem II by Miguel A. Hernandez-Prieto; Tania Tibiletti; Leyla Abasova; Diana Kirilovsky; Imre Vass; Christiane Funk (pp. 1143-1151).

The five small CAB-like proteins (ScpA-E) of the cyanobacterium Synechocystis sp. PCC 6803 belong to the family of stress-induced light-harvesting-like proteins, but are constitutively expressed in a mutant deficient of Photosystem I (PSI). Using absorption, fluorescence and thermoluminescence measurements this PSI-less strain was compared with a mutant, in which all SCPs were additionally deleted. Depletion of SCPs led to structural rearrangements in Photosystem II (PSII): less photosystems were assembled; and in these, the Q_B site was modified. Despite the lower amount of PSII, the SCP-deficient cells contained the same amount of phycobilisomes (PBS) as the control. Although the excess PBS were functionally disconnected, their fluorescence was quenched under high irradiance by the activated Orange Carotenoid Protein (OCP). Additionally the amount of OCP, but not of the iron-stress induced protein (isiA), was higher in this SCP-depleted mutant compared with the control. As previously described, the lack of SCPs affects the chlorophyll biosynthesis (Vavilin, D., Brune, D. C., Vermaas, W. (2005) Biochim Biophys Acta 1708, 91–101). We demonstrate that chlorophyll synthesis is required for efficient PSII repair and that it is partly impaired in the absence of SCPs. At the same time, the amount of chlorophyll also seems to influence the expression of ScpC and ScpD.► Using absorption, fluorescence and thermoluminescence measurements a PSI-less strain was compared with a mutant, in which all small CAB-like proteins (SCPs) were additionally deleted. ► Depletion of SCPs led to structural rearrangements in Photosystem II (PSII): less photosystems were assembled; and in these, the Q_B site was modified. ► Despite the lower amount of PSII, the SCP-deficient cells contained the same amount of phycobilisomes (PBS) as the control. A fraction of these PBS were functionally disconnected from PSII, but kept the capacity to perform non-photochemical quenching. ► Chlorophyll synthesis is required for efficient PSII repair, but that is partly impaired in the absence of the SCPs. The amount of chlorophyll also seems to influence the expression of ScpC and ScpD.

Keywords: Abbreviations; CAB; chlorophyll; a; /; b; binding; DCMU; 3-(3, 4-dichloro-phenyl)-1, 1-dimethylurea; Lil; light-harvesting-like proteins; OCP; orange carotenoid protein; PS; Photosystem; SCP; small CAB-like proteinsPhotosystem II; Non-photochemical quenching; Light-harvesting-like proteins; Synechocystis; sp. PCC 6803; High-light inducible proteins; Chlorophyll

Modeling Photosystem I with the alternative reaction center protein PsaB2 in the nitrogen fixing cyanobacterium Nostoc punctiforme by Ann Magnuson; Henning Krassen; Stensjo Karin Stensjö; Felix M. Ho; Stenbjörn Styring (pp. 1152-1161).

Five nitrogen fixing cyanobacterial strains have been found to contain PsaB2, an additional and divergent gene copy for the Photosystem I reaction center protein PsaB. In all five species the divergent gene, psaB2, is located separately from the normal psaAB operon in the genome. The protein, PsaB2, was recently identified in heterocysts of Nostoc punctiforme sp. strain PCC 73102. 12 conserved amino acid replacements and one insertion, were identified by a multiple sequence alignment of several PsaB2 and PsaB1 sequences. Several, including an inserted glutamine, are located close to the iron-sulfur cluster F_X in the electron transfer chain. By homology modeling, using the Photosystem I crystal structure as template, we have found that the amino acid composition in PsaB2 will introduce changes in critical parts of the Photosystem I protein structure. The changes are close to F_X and the phylloquinone (PhQ) in the B-branch, indicating that the electron transfer properties most likely will be affected. We suggest that the divergent PsaB2 protein produces an alternative Photosystem I reaction center with different structural and electron transfer properties. Some interesting physiologcial consequences that this can have for the function of Photosystem I in heterocysts, are discussed.► Five cyanobacterial species contain PsaB2, a divergent gene copy of the Photosystem I protein PsaB. ► PsaB2 is found in heterocysts of the Nostoc genus and may be involved in nitrogen fixation. ► Conserved amino acid replacements are found in PsaB2, in highly conserved regions close to F_X. ► The sequence of PsaB2 is likely to affect electron transfer on the acceptor side of Photosystem I.

Keywords: Abbreviations; ATP; adenosine triphosphate; Chl-; a; Chlorophyll-; a; NADPH; Nicotinamide adenine dinucleotide phosphate; PSI; Photosystem I; PSII; Photosystem IIPhotosystem-I; Nostoc punctiforme; Electron transfer; Homology model; Multiple sequence alignment

Time-resolved single-turnover of caa₃ oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts O_H into E_H state by Sergey A. Siletsky; Ilya Belevich; Nikolai P. Belevich; Tewfik Soulimane; Michael I. Verkhovsky (pp. 1162-1169).

The oxidative part of the catalytic cycle of the caa₃-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (CompoundsA,P,F) reproduce generally the features typical for the aa₃-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center—a heme center of the covalently bound cytochrome c. CompoundA was formed with significantly smaller yield compared to aa₃ oxidases in general and to ba₃ oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu_A and heme c are transferred in a single transition to the binuclear center during reduction of the compoundF, converting the binuclear center through the highly reactiveO_H state into the final product of the reaction—E_H (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa₃ -type enzymes, we concluded that the finally producedE_H state of caa₃ oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to theO_H→E_H transition of the aa₃-type oxidases. So, the fully-reduced caa₃ oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.► We performed kinetic study of electron and proton transfer in the 5 electron containing caa₃ oxidase ► For the decomposition of the molecular events spectra of the individual enzyme states were obtained ► Decomposition shows that additional fifth electron ends up in the catalytic center of the enzyme ► The delivery of the additional electron to the catalytic center is coupled to the proton pumping.

Keywords: Abbreviations; BNC; binuclear catalytic center; C; c; O; cytochrome; c; oxidase; RubiPy; tris(2,2′-bipyridyl) ruthenium; CHES; 2-(N-cyclohexylamino)ethane sulfonic acid; DM; n; -dodecyl-β-; d-; maltoside; MOPS; 3-(N-morpholino)propanesulfonic acid; TMPD; N,N,N′,N′-tetramethyl-1,4-phenylenediamine; τ; time constant of a processCatalytic cycle intermediates; Cytochrome; c; oxidase; Electron transfer; Thermus thermophilus

Bis-THF motif of acetogenin binds to the third matrix-side loop of ND1 subunit in mitochondrial NADH-ubiquinone oxidoreductase by Sayo Nakanishi; Masato Abe; Shuhei Yamamoto; Masatoshi Murai; Hideto Miyoshi (pp. 1170-1176).

Natural acetogenins are among the most potent inhibitors of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I). Our photoaffinity labeling study suggested that the hydroxylated bis-THF ring moiety of acetogenins binds at “site A” in the third matrix-side loop connecting the fifth and sixth transmembrane helices in the ND1 subunit [Kakutani et al. (2010) Biochemistry 49, 4794–4803]. Nevertheless, since this proposition was led using a photoreactive Δlac-acetogenin derivative, it needs to be directly verified using a natural acetogenin-type probe. We therefore conducted photoaffinity labeling using a photoreactive natural acetogenin mimic ([¹²⁵I] di azinylated natural acetogenin, [¹²⁵I]DANA), which has a small photolabile diazirine group, in place of a hydroxy group, attached to the bis-THF ring moiety. Analysis of the photocross-linked protein in bovine heart submitochondrial particles unambiguously revealed that [¹²⁵I]DANA binds to the membrane subunit ND1 with high specificity. The photocross-linking was completely blocked in the presence of just a 5-fold excess of bullatacin, indicating that [¹²⁵I]DANA is an excellent mimic of natural acetogenins and hence binds to the site that accommodates natural products. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by different proteases and their combinations indicated that the cross-linked residue is predominantly located at the supposed site A in the third matrix-side loop.Display Omitted► We synthesized an acetogenin mimic having photoreactive group in bis-THF ring. ► We performed photoaffinity labeling to identify the binding site of bis-THF motif. ► Bis-THF motif of acetogenins binds to the ND1 subunit of bovine complex I. ► The binding site is located in the third matrix-side loop of ND1 subunit.

Keywords: Abbreviations; BN-PAGE; blue native polyacrylamide gel electrophoresis; CBB; coomassie brilliant blue; complex I; mitochondrial proton-pumping NADH-ubiquinone oxidoreductase; DAA; diazirinylated Δlac-acetogenin; [; ¹²⁵; I]DAA; ¹²⁵; I-labeled DAA; DANA; diazinylated natural acetogenin; [; ¹²⁵; I]DANA; ¹²⁵; I-labeled DANA; DDM; n; -dodecyl β-D-maltoside; IC; ₅₀; the molar concentration (nM) needed to reduce the control NADH oxidase activity in SMP by half; LHON; Leber's hereditary optic neuropathy; MELAS; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes; MALDI-TOF; matrix-assisted laser desorption ionization time of flight; MS; mass spectrometry; PVDF; polyvinylidene fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SMP; submitochondrial particles; THF; tetrahydrofuranMitochondrial complex I; Acetogenin; Respiratory inhibitor; Photoaffinity labeling; ND1 subunit

Moderate heat stress induces state transitions in Arabidopsis thaliana by Sreedhar Nellaepalli; Nageswara Rao Mekala; Otto Zsiros; Prasanna Mohanty; Rajagopal Subramanyam (pp. 1177-1184).

The effect of temperature on the photosynthetic machinery is crucial for the fundamental understanding of plant physiology and the bioengineering of heat-tolerant varieties. In our study, Arabidopsis thaliana was exposed to mild (40°C), short-term heat stress in the dark to evaluate the heat-triggered phosphorylation and migration of light harvesting complex (LHC) II in both wild-type (wt) and mutant lacking STN7 kinase. The 77K emission spectra revealed an increase in PSI relative to PSII emission similar to increases observed in light-induced state I to state II transitions in wt but not in stn7 mutant. Immunoblotting results indicated that the major LHCII was phosphorylated at threonine sites under heat stress in wt plants but not in the mutant. These results support the proposition that mild heat stress triggers state transitions in the dark similar to light-induced state transitions, which involve phosphorylation of LHCII by STN7 kinase. Pre-treatment of Arabidopsis leaves with inhibitor DBMIB, altered the extent of LHCII phosphorylation and PSI fluorescence emission suggests that activation of STN7 kinase may be dependent on Cyt b₆/f under elevated temperatures in dark. Furthermore, fast Chl a transient of temperature-exposed leaves of wt showed a decrease in the F_v/ F_m ratio due to both an increase in F_o and a decrease in F_m. In summary, our findings indicate that a mild heat treatment (40°C) induces state transitions in the dark resulting in the migration of phosphorylated LHCII from the grana to the stroma region.► Elevated temperature can induce phosphorylation of LHCII. ► The STN7 kinase is responsible for LHCII phosphorylation. ► STN7 kinase activation was dependent of the redox state of electron carriers. ► Temperature induced state transition is similar to light induced state transition.

Keywords: Abbreviations; Cyt b; ₆; /f; cytochrome b; ₆; /f; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB; 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; LHCII; light harvesting complex II; OEC; oxygen evolving complex; PEA; plant efficiency analyzer; PQ pool; plastoquinone; PS; photosystems; RC; reaction center; wt; wild-type Arabidopsis thaliana; Elevated temperature; Photosystems; PQ pool; State transition; STN7 kinase

ATP synthase superassemblies in animals and plants: Two or more are better by Holger Seelert; Norbert A. Dencher (pp. 1185-1197).

ATP synthases are part of the sophisticated cellular metabolic network and therefore multiple interactions have to be considered. As discussed in this review, ATP synthases form various supramolecular structures. These include dimers and homooligomeric species. But also interactions with other proteins, particularly those involved in energy conversion exist. The supramolecular assembly of the ATP synthase affects metabolism, organellar structure, diseases, ageing and vice versa. The most common approaches to isolate supercomplexes from native membranes by use of native electrophoresis or density gradients are introduced. On the one hand, isolated ATP synthase dimers and oligomers are employed for structural studies and elucidation of specific protein–protein interactions. On the other hand, native electrophoresis and other techniques serve as tool to trace changes of the supramolecular organisation depending on metabolic alterations. Upon analysing the structure, dimer-specific subunits can be identified as well as interactions with other proteins, for example, the adenine nucleotide translocator. In the organellar context, ATP synthase dimers and oligomers are involved in the formation of mitochondrial cristae. As a consequence, changes in the amount of such supercomplexes affect mitochondrial structure and function. Alterations in the cellular power plant have a strong impact on energy metabolism and ultimately play a significant role in pathophysiology. In plant systems, dimers of the ATP synthase have been also identified in chloroplasts. Similar to mammals, a correlation between metabolic changes and the amount of the chloroplast ATP synthase dimers exists. Therefore, this review focusses on the interplay between metabolism and supramolecular organisation of ATP synthase in different organisms.► ATP synthases form dimers and homooligomeric species, but also interact with other proteins. ► Supramolecular assembly affects metabolism, organelles, diseases, ageing, and vice versa. ► Ways to isolate native supercomplexes and study supramolecular organisation are introduced. ► In mitochondria, ATP synthases are involved in the formation of cristae and interact with the ANT. ► A correlation between metabolic changes and the amount of the ATP synthase dimers exists.

Keywords: Abbreviations; ANT; adenine nucleotide translocator; ASA; ATP synthase associated; BHM; bovine heart mitochondria; BN; blue-native; CBBG-250; Coomassie Brilliant Blue G-250; CF; ₁; F; _o; chloroplast ATP synthase; (C)F; _o; membrane integral part of the (chloroplast) ATP synthase; CN; colourless native/clear native; DCCD; N; ,; N; ′-dicyclohexylcarbodiimide; F; ₁; hydrophilic part of the ATP synthase; OxPhos; oxidative phosphorylationOxPhos supercomplexes; Native electrophoresis; F; ₁; F; _o; ATP synthasome; Solubilisation

Human diseases with impaired mitochondrial protein synthesis by Rotig Agnès Rötig (pp. 1198-1205).

Mitochondrial respiratory chain deficiencies represent one of the major causes of metabolic disorders that are related to genetic defects in mitochondrial or nuclear DNA. The mitochondrial protein synthesis allows the synthesis of the 13 respiratory chain subunits encoded by mtDNA. Altogether, about 100 different proteins are involved in the translation of the 13 proteins encoded by the mitochondrial genome emphasizing the considerable investment required to maintain mitochondrial genetic system. Mitochondrial protein synthesis deficiency can be caused by mutations in any component of the translation apparatus including tRNA, rRNA and proteins. Mutations in mitochondrial rRNA and tRNAs have been first identified in various forms of mitochondrial disorders. Moreover abnormal translation due to mutation in nuclear genes encoding tRNA-modifying enzymes, ribosomal proteins, aminoacyl-tRNA synthetases, elongation and termination factors and translational activators have been successively described. These deficiencies are characterized by a huge clinical and genetic heterogeneity hampering to establish genotype–phenotype correlations and an easy diagnosis. One can hypothesize that a new technique for gene identification, such as exome sequencing will rapidly allow to expand the list of genes involved in abnormal mitochondrial protein synthesis.► This review presents the human diseases associated with mitochondrial translation deficiency. ► The mt translation allows the synthesis of the 13 proteins encoded by mtDNA. ► Translation deficiencies are caused by mutations in various tRNA, rRNA and proteins. ► The clinical presentation and the genetic causes are extremely heterogeneous.

Keywords: Abbreviations; AARS; aminoacyl-tRNA synthetase; LSFC; Leigh Syndrome French Canadian variant; MLASA; mitochondrial myopathy, lactic acidosis and sideroblastic anemia; MRP; mitochondrial ribosomal proteins; mt; mitochondrial; mtDNA; mitochondrial DNA; OXPHOS; oxidative phosphorylation; PEO; progressive external ophthalmoplegia; RC; respiratory chain; rRNA; ribosomal RNA; SNHL; sensorineural hearing loss; tRNA; transfer RNAMitochondria; Translation; Genetic disease

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid by Tatjana M. Hildebrandt (pp. 1206-1213).

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H₂S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.► Dehydroascorbic acid decreases sulfide toxicity in rat mitochondria. ► Dehydroascorbic acid increases rates of sulfide oxidation. ► Inhibition of cytochrome c oxidase by sulfide can be modulated. ► The protective effect requires supermolecular association of cytochrome c oxidase.

Keywords: Abbreviations; DHA; dehydroascorbic acid; TMPD; N,N,N′,N′-tetramethyl-p-phenylenediamine; I; _max; maximum inhibitory effectAscorbic acid; Enzyme inhibitor; Mitochondrial metabolism; Respiratory chain; Sulfide

Herbicide effect on the photodamage process of photosystem II: Fourier transform infrared study by Issei Idedan; Tatsuya Tomo; Takumi Noguchi (pp. 1214-1220).

The photodamage process of photosystem II by strong illumination was investigated by examining the herbicide effects on the photoinactivation of redox cofactors. O₂-evolving photosystem II membranes from spinach in the absence of herbicide and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and bromoxynil were subjected to strong white-light illumination at 298K, and the illumination-time dependence of the activities of Q_A, the Mn cluster, and P680 were monitored using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The decrease in the Q_A activity was suppressed and accelerated by DCMU and bromoxynil, respectively, in comparison with the sample without herbicide. The intensity change in the S₂/S₁ FTIR signal of the Mn cluster exhibited a time course virtually identical to that in the Q_A signal in all the three samples, suggesting that the loss of the S₁→S₂ transition was ascribed to the Q_A inactivation and hence the Mn cluster was inactivated not faster than Q_A. The decrease in the P680 signal was always slower than that of Q_A keeping the tendency of the herbicide effect. Degradation of the D1 protein occurred after the P680 inactivation. These observations are consistent with the acceptor-side mechanism, in which double reduction of Q_A triggers the formation of¹O₂* to promote further damage to other cofactors and the D1 protein, rather than the recently proposed mechanism that inactivation of the Mn cluster initiates the photodamage. Thus, the results of the present study support the view that the acceptor-side mechanism dominantly occurs in the photodamage to PSII by strong white-light illumination.► The photodamage process of PSII was investigated using FTIR spectroscopy. ► The damage to Q_A was suppressed by DCMU and accelerated by bromoxynil. ► The Mn cluster was inactivated not faster than Q_A. ► The decrease in the P680 signal was always slower than Q_A. ► The results support the acceptor-side mechanism in the PSII photodamage.

Keywords: Abbreviations; Chl; _D1; monomeric chlorophyll on the D1 side of PSII; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; FTIR; Fourier transform infrared; Mes; 2-(; N; -morpholino)ethanesulfonic acid; P680; special pair chlorophylls in PSII; Pheo; redox-active pheophytin in PSII; PQH; ₂; plastoquinol; PSII; photosystem II; Q; _A; primary quinone electron acceptor; Q; _B; secondary quinone electron acceptorFTIR; Herbicide; Photodamage; Photoinhibition; Photoprotection; Photosystem II

Effects of cyclosis on chloroplast–cytoplasm interactions revealed with localized lighting in Characean cells at rest and after electrical excitation by Alexander A. Bulychev; Svetlana O. Dodonova (pp. 1221-1230).

Cytoplasmic streaming in Characean internodes enables rapid intracellular transport and facilitates interactions between spatially remote cell regions. Cyclosis-mediated distant interactions might be particularly noticeable under nonuniform illumination, in the vicinity of light–shade borders where metabolites are transported between functionally distinct cell regions. In support of this notion, chlorophyll fluorescence parameters assessed on a microscopic area of Chara corallina internodal cells (area of inspection, AOI) responded to illumination of nearby regions in asymmetric manner depending on the vector of cytoplasmic streaming. When a beam of white light was applied through a 400-μm optic fiber upstream of AOI with regard to the direction of cytoplasmic streaming, non-photochemical quenching (NPQ) developed after a lag period in AOI exposed to moderate intensity light. Conversely, no NPQ was induced in the same cell area when the beam position was shifted to an equal distance downstream of AOI. Light–response curves for the efficiency of photosystem II electron transport in chloroplasts differed markedly depending on the illumination pattern (whole-cell versus small area illumination) but these differences were eliminated after the inhibition of cytoplasmic streaming with cytochalasin B. Localized illumination promoted chloroplast fluorescence responses to electrical plasmalemma excitation at high light intensities, which contrasts to the requirement of low to moderate irradiances for observation of the stimulus–response coupling under whole-cell illumination. The results indicate that different photosynthetic capacities of chloroplasts under general and localized illumination are related to lateral transport of nonevenly distributed cytoplasmic components between the cell parts with dominant photosynthetic and respiratory metabolism.► Cyclosis provides functional polarity to cell areas around a light spot in Chara. ► A mediator of interplastid interactions moves at a velocity of streaming cytoplasm. ► Local cell fluorescence changes may arise after and aside from incident light pulse. ► Cytoplasm inflow from shaded areas elevates photosynthetic capacity of chloroplasts. ► Light pattern affects the cell fluorescence response to membrane electrical excitation.

Keywords: Abbreviations; AOI; area of inspection (area of fluorescence measurements in the chloroplast layer of; Chara; cell); NPQ; non-photochemical quenching; PAR; photosynthetically active radiation; PFD; photon flux density; PSII; photosystem II Chara corallina; Cytoplasmic streaming; Localized illumination; Non-photochemical quenching; Cytochalasin B; Action potential

Efficiency of photosynthesis in a Chl d-utilizing cyanobacterium is comparable to or higher than that in Chl a-utilizing oxygenic species by S.P. Mielke; N.Y. Kiang; R.E. Blankenship; M.R. Gunner; D. Mauzerall (pp. 1231-1236).

The cyanobacterium Acaryochloris marina uses chlorophyll d to carry out oxygenic photosynthesis in environments depleted in visible and enhanced in lower-energy, far-red light. However, the extent to which low photon energies limit the efficiency of oxygenic photochemistry in A. marina is not known. Here, we report the first direct measurements of the energy-storage efficiency of the photosynthetic light reactions in A. marina whole cells, and find it is comparable to or higher than that in typical, chlorophyll a-utilizing oxygenic species. This finding indicates that oxygenic photosynthesis is not fundamentally limited at the photon energies employed by A. marina, and therefore is potentially viable in even longer-wavelength light environments.► The energy-storage efficiency of photosynthesis in A. marina is found to be 40±1%. ► The energy-storage efficiency in S. leopoliensis is found to be 34±1%. ► These results are consistent with theoretical estimates of the maximum efficiency. ► Mechanisms of photochemistry in A. marina are discussed in terms of the Z-scheme.

Keywords: Acaryochloris marina; Oxygenic photosynthesis; Energy-storage efficiency; Photoacoustics

Light-induced isomerization of the LHCII-bound xanthophyll neoxanthin: Possible implications for photoprotection in plants by Monika Zubik; Rafał Luchowski; Wojciech Grudzinski; Małgorzata Gospodarek; Ignacy Gryczynski; Zygmunt Gryczynski; Jerzy W. Dobrucki; Wiesław I. Gruszecki (pp. 1237-1243).

Light-harvesting pigment-protein complex of Photosystem II (LHCII) is the largest photosynthetic antenna complex of plants and the most abundant membrane protein in the biosphere. Plant fitness and productivity depend directly on a balance between excitations in the photosynthetic apparatus, generated by captured light quanta, and the rate of photochemical processes. Excess excitation energy leads to oxidative damage of the photosynthetic apparatus and entire organism and therefore the balance between the excitation density and photosynthesis requires precise and efficient regulation, operating also at the level of antenna complexes. We show that illumination of the isolated LHCII leads to isomerization of the protein-bound neoxanthin from conformation 9′- cis to 9′,13- and 9′,13′- dicis forms. At the same time light-driven excitation quenching is observed, manifested by a decrease in chlorophyll a fluorescence intensity and shortened fluorescence lifetimes. Both processes, the neoxanthin isomerization and the chlorophyll excitation quenching, are reversible in dim light. The results of the 77K florescence measurements of LHCII show that illumination is associated with appearance of the low-energy states, which can serve as energy traps in the pigment–protein complex subjected to excess excitation. Possible sequence of the molecular events is proposed, leading to a protective excess excitation energy quenching: neoxanthin photo-isomerization→formation of LHCII supramolecular structures which potentiate creation of energy traps→excitation quenching.Display Omitted► Photo-isomerization of neoxanthin bound to LHCII is observed. ► Illumination of LHCII with blue light induces excitation quenching. ► Light-induced excitation quenching in LHCII is associated with appearance of low-energy states (traps).

Keywords: Photosynthesis; Photoprotection; Xanthophyll; Spectroscopy; Fluorescence; LHCII

The role of pH on the thermodynamics and kinetics of muscle biochemistry: An in vivo study by³¹P-MRS in patients with myo-phosphorylase deficiency by E. Malucelli; S. Iotti; D.N. Manners; C. Testa; A. Martinuzzi; B. Barbiroli; R. Lodi (pp. 1244-1249).

In this study we assessed ΔG′_ATP hydrolysis, cytosolic [ADP], and the rate of phosphocreatine recovery using Phosphorus Magnetic Resonance Spectroscopy in the calf muscle of a group of patients affected by glycogen myo-phosphorylase deficiency (McArdle disease). The goal was to ascertain whether and to what extent the deficit of the glycogenolytic pathway would affect the muscle energy balance. A typical feature of this pathology is the lack of intracellular acidosis. Therefore we posed the question of whether, in the absence of pH decrease, the rate of phosphocreatine recovery depends on the amount of phosphocreatine consumed during exercise. Results showed that at the end of exercise both [ADP] and ΔG′_ATP of patients were significantly higher than those of matched control groups reaching comparable levels of phosphocreatine concentration. Furthermore, in these patients we found that the rate of phosphocreatine recovery is not influenced by the amount of phosphocreatine consumed during exercise. These outcomes provide experimental evidence that: i) the intracellular acidification occurring in exercising skeletal muscle is a protective factor for the energy consumption; and ii) the influence of pH on the phosphocreatine recovery rate is at least in part related to the kinetic mechanisms of mitochondrial creatine kinase enzyme.► Intracellular acidification increases the thermodynamic efficiency of ATP hydrolysis. ► At higher energy demand “acidification protective effect” is more pronounced. ► The rate of PCr recovery only depends on cytosolic pH. ► The rate of PCr recovery is not influenced by the amount of PCr consumed.

Keywords: Abbreviations; ³¹; P-MRS; Phosphorus Magnetic Resonance Spectroscopy; PCr; phosphocreatine; MCK; mitochondrial creatine kinase enzyme; Cr; creatine; TR; repetition time; FWMH; Full Widths at Middle Height; [PCr]; _cons; PCr consumed during exercise; TC; _PCr; PCr recovery time constant; Vi; initial rate of PCr recovery; L; light exercise protocol; M; moderate exercise protocol; LBM; lean body massSkeletal muscle; Glycogen; Energy metabolism; Phosphorous magnetic resonance spectroscopy; McArdle disease; ΔG ATP

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BBA - Bioenergetics (v.1807, #9)

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BBA - Bioenergetics (v.1807, #9)